Genetic Diversity and Natural Selection of the Plasmodium knowlesi Circumsporozoite Protein Nonrepeat Regions

Background

Plasmodium knowlesi is a simian malaria parasite that has been identified to cause malaria in humans. To date, several thousand cases of human knowlesi malaria have been reported around Southeast Asia. Thus far, there is no detailed study on genetic diversity and natural selection of P. knowlesi circumsporozoite protein (CSP), a prominent surface antigen on the sporozoite of the parasite. In the present study, the genetic diversity and natural selection acting on the nonrepeat regions of the gene encoding P. knowlesi CSP were investigated, focusing on the T-cell epitope regions at the C-terminal of the protein.

Methods

Blood samples from 32 knowlesi malaria patients and 2 wild monkeys (Macaca fascicularis) were used. The CSP of the P. knowlesi isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The nonrepeat regions of the CSP gene were analysed for genetic diversity, natural selection and haplotypic grouping using MEGA5 and DnaSP version 5.10.00 programmes. A haplotype network was constructed based on the C-terminal (Th2R/Th3R) T-cell epitope regions using the Median-Joining method in the NETWORK version 4.6.1.2 programme. Previously published sequences from other regions (Malaysia Borneo, Singapore) were also included in the analysis.

Results

A total of 123 P. knowlesi CSP sequences were analysed. Multiple sequence alignment revealed 58 amino acid changes, and 42 novel amino acid haplotypes were identified. Polymorphism was higher in the C-terminal Th2R/Th3R epitope (π = 0.0293, n = 123) region compared to the overall combined nonrepeat regions (π = 0.0120, n = 123). Negative natural selection was observed within the nonrepeat regions of the CSP gene. Within the C-terminal Th2R/Th3R epitope regions, there was evidence of slight positive selection. Based on haplotype network analysis of the Th2R/Th3R regions, five abundant haplotypes were identified. Sharing of haplotypes between humans and macaques were observed.

Conclusion

This study contributes to the understanding of the type and distribution of naturally occurring polymorphism in the P. knowlesi CSP gene. This study also provides a measurement of the genetic diversity of P. knowlesi and identifies the predominant haplotypes within Malaysia based on the C-terminal Th2R/Th3R regions.     

IL-15 and macrophage secretory factors facilitate immune activation of neonatal natural killer cells by lipoteichoic acid

Neonates possess a relatively “naive”, yet inducible immune system. Our hypothesis is that upon strategic antigen exposure, cytokine priming and sensitization by accessory cells, natural killer (NK) cells could be activated to become a functional phenotype. We investigated the in vitro stimulation of cord blood (CB) and adult NK cells upon challenge with lipoteichoic acid (LTA), interleukin (IL)-15 and LTA-primed autologous macrophage-conditioned medium, using CD107a and CD69 phenotypes as indicators of activation. We also examined response of CB macrophages to LTA, in terms of P44/42 extracellular signal-regulated kinases (ERK1/2) activation and cytokine secretion. LTA significantly induced secretion of inflammatory cytokines tumor necrotic factor (TNF)-α, IL-6, IL-12 and activated the upstream signal of ERK1/2 phosphorylation in neonatal macrophages. The magnitude of responses to stimulation differed between neonatal and adult NK cells. Co-stimulation with IL-15 was critical for expansion of the CD69 and CD107a NK subpopulations in both neonatal and adult cells, upon a LTA challenge. NK cell activation could be enhanced by LTA-primed autologous macrophages through secretory factors. Our results indicated that neonatal macrophages and NK cells can evoke immunologic responses to a Gram-positive bacterial antigen. The combinatory priming strategy is relevant for development of novel protocols, such as IL-15 treatment, to compensate for the immaturity of the innate immune system in newborns against bacterial infections.     

Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures

Since April 2012, there have been 17 laboratory-confirmed human cases of respiratory disease associated with newly recognized human betacoronavirus lineage C virus EMC (HCoV-EMC), and 7 of them were fatal. The transmissibility and pathogenesis of HCoV-EMC remain poorly understood, and elucidating its cellular tropism in human respiratory tissues will provide mechanistic insights into the key cellular targets for virus propagation and spread. We utilized ex vivo cultures of human bronchial and lung tissue specimens to investigate the tissue tropism and virus replication kinetics following experimental infection with HCoV-EMC compared with those following infection with human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus (SARS-CoV). The innate immune responses elicited by HCoV-EMC were also investigated. HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. While SARS-CoV productively replicated in lung tissue, replication in human bronchial tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there was minimal HCoV-229E infection in these tissues. HCoV-EMC failed to elicit strong type I or III interferon (IFN) or proinflammatory innate immune responses in ex vivo respiratory tissue cultures. Treatment of human lung tissue ex vivo organ cultures with type I IFNs (alpha and beta IFNs) at 1 h postinfection reduced the replication of HCoV-EMC, suggesting a potential therapeutic use of IFNs for treatment of human infection.     

Revising the ethnodevelopment model: addressing Karen self-determination within the context of the failed ethnocratic state of military-ruled Burma

My paper examines the Karen ethnic nationality and their fifty-eight-year self-determination struggle against ethnic cleansing resulting from the ethnocratic and military governments of Burma. I frame Karen self-determination as a development issue by employing Rodolfo Stavenhagen's ethnodevelopment model. Ethnodevelopment argues that, if asymmetrical development occurs within a multi-ethnic state, state-oriented ethnic minority development strategies are needed to neutralize the asymmetry. However, Stavenhagen's ethnodevelopment does not question the premise of an authoritarian state or the systemic crisis experienced by ethnic minorities under authoritarian rule. Thus, I revise ethnodevelopment from its top-to-bottom trajectory where ethnic minority development is dependent upon the centralized state, to a bottom-to-top trajectory I designate as liberation ethnodevelopment. I argue that Karen liberation ethnodevelopment is also a development process, but one that develops and shields the Karen from ethnic cleansing.     

Human Wharton's Jelly stem cell conditioned medium and cell‐free lysate inhibit human osteosarcoma and mammary carcinoma cell growth in vitro and in xenograft mice

Human Wharton's jelly stem cells (hWJSCs) were shown to inhibit the growth of human mammary carcinomas. It is not known whether cell‐free secretions or lysates of hWJSCs do the same on different cancers. They may be less controversial than cells to regulatory bodies for clinical application. We examined the influence of hWJSC conditioned medium (hWJSC‐CM) and cell‐free lysate (hWJSC‐CL) on two osteosarcoma cell lines (MG‐63, SKES‐1) in vitro and on human mammary carcinomas in immunodeficient mice. When exposed to hWJSC‐CL, increased vacuolations in MG‐63 and increased membrane fragmentation in SKES‐1 cells were observed, with greater cell death in SKES‐1. Exposure of SKES‐1 and MG‐63 cells to hWJSC‐CL showed significant decreases in cell proliferation of 46.48 ± 6.66% and 24.32 ± 5.67% respectively compared to controls. MG‐63 and SKES‐1 cells were annexin V‐FITC positive and SKES‐1 TUNEL positive following treatment with hWJSC‐CM and hWJSC‐CL. MG‐63 cells were positive and SKES‐1 cells negative for anti‐BECLIN‐1 and anti‐LC3B following treatment with hWJSC‐CM and hWJSC‐CL. RT‐PCR showed that the pro‐apoptotic BAX gene and the autophagy‐related ATG‐5 and BECLIN‐1 genes were up‐regulated while the anti‐apoptotic BCL2 and SURVIVIN genes were down‐regulated in MG‐63 and SKES‐1 cells treated with hWJSC‐CM and hWJSC‐CL. Injections of hWJSCs and hWJSC‐CM into mammary carcinomas in immunodeficient mice resulted in decreased tumor sizes and weights of 24.86 ± 6.05% to 37.03 ± 5.91% and 47.14 ± 7.36% to 55.09 ± 5.87% respectively at 6 weeks compared to controls. hWJSC‐CM and hWJSC‐CL inhibit mammary carcinoma and osteosarcoma cells via apoptosis and autophagy. J. Cell. Biochem. 114: 366–377, 2013. © 2012 Wiley Periodicals, Inc.     

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the